The bifunctional dihydrofolate reductase thymidylate synthase of Tetrahymena thermophila provides a toll for molecular and biotechnology applications

Abstract

Background: Dihydrofolate reductase (DHFR) and thymidylate synthase (TS) are crucial enzymes in DNA synthesis. In alveolata both enzymes are expressed as one bifunctional enzyme. Results: Loss of this essential enzyme activities after successful allelic assortment of knock out alleles yields an auxotrophic marker in ciliates. Here the cloning, characterisation and functional analysis of Tetrahymena thermophila's DHFR-TS is presented. A first aspect of the presented work relates to destruction of DHFR-TS enzyme function in an alveolate thereby causing an auxotrophy for thymidine. A second aspect is to knock in an expression cassette encoding for a foreign gene with subsequent expression of the target protein. Conclusion: This system avoids the use of antibiotics or other drugs and therefore is of high interest for biotechnological applications. © 2006 Herrmann et al; licensee BioMed Central Ltd.